The broad goal of this proposal is to characterize novel protein-protein interactions involving the inositol trisphosphate receptor (InsP3R) Ca 2+release channel that we have identified. The InsP3R participates in generation of complex Ca 2+signals that regulate many physiological processes. Interactions between the InsP3R and protein components of signaling pathways, cytoskeleton or membranes could provide effective means to couple Ca 2+release to specific targets, or to regulate the Ca 2vrelease properties of the channel. We recently identified CaBPs, a family of proteins in the neuronal Ca 2+sensor sub-family of calmodulin related proteins, as high-affinity interactors with the InsP3R. Importantly, binding of CaBPs to the InsP3- binding region activates gating of the channel in the absence of InsP3, thereby identifying CaBPs as protein ligands of the InsP3R. Because InsP3R-mediated Ca 2+ signals are shaped by messenger diffusion, degradation and removal, processes that will have distinct kinetics for InsP3 compared with CaBPs, our identification of novel ligands for the InsP3R provides new insights into the dimensions and versatility of this ubiquitous signaling pathway. We propose three specific aims to characterize the mechanisms and function of the interaction between the InsP3R and CaBPs. First, we will define the mechanisms of CaBP gating of the channel using a combination of biochemical approaches and single channel recording of endogenous Xenopus and recombinant mammalian InsP3R channels in their native endoplasmic reticulum membrane in oocyte nuclear envelopes. Second, we will determine structural bases for the interaction, using mutagenesis and peptide competition approaches. Here, we will also examine interactions of other CaBP proteins with the InsP3R, and determine whether other NCBP proteins interact with and gate the channel, using electrophysiological and biochemical approaches. Third, we will determine the physiological roles of the interaction of CaBPs with the InsP3R. We will examine interactions in cells, using biochemical and optical approaches, and test two hypotheses in cellular models. First, that the interaction provides a mechanism to titrate sensitivity of the channel to InsP3. Second, that the interaction enables the channel to generate Ca 2+ signals in the absence of engagement of the phosphoinositide system. The results of these studies should provide new insights into a novel concept in the regulation of Ca 2 release.